Transcript Paroxysmal Nocturnal Hemoglobinuria March 8, 2005 Case       43 y old Hispanic man who presented to his PCP for headaches.

Paroxysmal Nocturnal
Hemoglobinuria
March 8, 2005
Case
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43 y old Hispanic man who presented to his PCP for
headaches. Labs revealed a pancytopenia. Referred to
hematologist.
Bone marrow revealed hypocellular marrow (5-10%).
Bone marrow repeated 6 months later showed minimally
hypocellular marrow (30%).
Two years later, he developed dark urine and hemolytic
anemia.
Bone marrow showed hypercellularity (80%) with
normoblastic erythroid hyperplasia. A significant population of
myeloid cells (85%) demonstrated atypically diminished
expression of CD16, as well as an aberrant lack of CD55 and
CD59 expression. A significant population of monocytic cells
(83%) lacked expression of CD14, CD55, and CD59.
Findings consistent with PNH.
History
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Investigator Year
Gull
1866
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Strubing
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1882
van den Burgh 1911
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Enneking
1928
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Marchiafava
and Micheli
19281931
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Ham
19371939
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Davitz
1986
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Hall & Rosse
1996
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Contribution
Described nocturnal and paroxysmal nature of
“intermittent haematinuria” in a young man.
Distinguished PNH from paroxysmal cold
haemoglobinuria and march haemoglobinuria.
Attributed the problem to the red cells.
Red cells lysed in acidified serum. Suggested a role
for complement.
Coined the name “paroxysmal nocturnal
haemoglobinuria”.
Described perpetual hemosiderinemia.
Their names became eponymous for PNH
in Europe.
Identified the role of complement in lysis of PNH red
cells. Developed the acidified serum test, also called
the Ham test, which is still used to diagnose PNH.
Demonstrated that only a portion of PNH red cells are
abnormally sensitive to complement.
Suggests defect in membrane protein anchoring
system responsible
Flow cytometry for the diagnosis of PNH
Paroxysmal Nocturnal
Hemoglobinuria
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Described as a clinical entity in 1882.
Acquired disorder of hematopoiesis.
Triad: intravascular hemolysis, thrombosis, and
decreased hematopoiesis.
“Nocturnal” refers to belief that hemolysis is triggered by
acidosis during sleep and activation of complement to
hemolyze abnormal RBCs.
However, hemolysis is shown to occur throughout the
day and is not paroxysmal. Urine concentrated overnight
may cause dramatic change in color.
Paroxysmal Nocturnal
Hemoglobinuria
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Due to an acquired hematopoietic stem cell mutation defect.
Somatic mutation (from deletions to point mutations identified)
of the PIGA (phosphotidyl-inositol glycan class A) gene on the
X-chromosome. Namely the transfer of N-acetylglucosamine
to phosphatidylinositol.
Hillmen and Richards, Br J Haematol, 2000
Paroxysmal Nocturnal
Hemoglobinuria
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Cells derived from the abnormal clone deficient in surface
proteins normally attached to the cell membrane by a
glycosylphoshpatidylinositol (GPI) anchor.
Essential group of membrane proteins lacking are called
complement regulating surface proteins – decay accelerating
factor (DAF) or CD55, homologous restriction factor (HRF) or C8
binding protein, and membrane inhibitor of reactive lysis (MIRL)
or CD59.
These proteins interact with complement proteins and interfere
with the assembly of complement's membrane-attack complex.
Deficiency CD59 is largely responsible for the hemolysis and
implicated in the thrombotic tendency (induces platelet
activation).
GPI Linked Proteins
Rosti, Haematologica, 2000
GPI anchored Proteins
Johnson and Hillmen,Mol Pathol, 2002
Paroxysmal Nocturnal
Hemoglobinuria
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Frequency: rate determined to be 5-10x less than
aplastic anemia (2/million). Perhaps more frequent
in Southeast Asia and Far East.
Men and women affected equally.
At any age, but frequently found among young
adults.
Mortality/Morbidity: median survival of 10.3 yrs.
Morbidity depends on variable expression of
hemolysis, bone marrow failure, and thrombophilia.
Main cause of death is venous thrombosis followed
by complications of bone marrow failure.
Hemolysis
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Hemoglobinuria/hemosiderinuria.
Intravascular hemolysis - elevated retic count and LDH with
low haptoglobin in the absence of hepatosplenomegaly.
Hemolytic anemia of variable severity: proportion of abnormal
cells, degree of abnormality of the cells, degree of
complement activation (viral or bacterial infections).
Bone marrow usually markedly erythroid with decreased iron
stores.
Can be precipitated by administration of Fe to an Fe deficient
patient due to large number of complement sensitive cells
delivered to circulation at once.
Thrombosis
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Hepatic vein (Budd-Chiari syndrome) – jaundice,
abdominal pain, hepatomegaly, ascites.
Abdominal vein thrombosis – can lead to bowel
infarction.
Cerebral vein thrombosis – if sagittal vein affected can
lead to papilledema and pseudotumor cerebri.
Dermal vein thrombosis – raised, painful, and red
nodules in skin affecting large areas.
Increased platelet aggregation, enhanced expression of
tissue factor, and impaired fibrinolysis.
In two series, almost all patients developing thrombosis
had more than 50% and 61% PNH granulocytes.
Cytopenias
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Deficient hematopoiesis
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Usually presents with anemia despite the presence of
an erythroid marrow with suboptimal reticulocytosis.
Neutropenia and thrombcytopenia can occur in a
hypoplastic bone marrow.
Other
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Esophageal spasms can occur concurrently with
episodes of hemoglobinuria. Manometry shows
generation of peristaltic waves of great intensity.
Males can have impotence.
Absence of nitric oxide (taken up by hemoglobin in
plasma)?
Abnormal Cells
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PNH I cells normal in sensitivity to complement.
PNH II cells moderately more sensitive than
normal cells (partial absence).
PNH III cells markedly sensitive, requiring one
fifteenth to one twentieth of complement for an
equal degree of lysis (complete absence). This
group is increased in patients with more severe
PNH and is associated with a mean life span of
10-15 days.
Diagnostic Test
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Acid hemolysis (Ham test): PNH red cells incubated in
separate tubes to fresh acidified serum (0.5mL),
unacidified serum, and heated acidified serum. Lysis
determined by optical density of the supernatant fluid
after 1hr incubation and addition of 4mL of 0.15 M NaCl.
Positive test >1% lysis in acidified serum.
May be positive in congenitial dyserythro-poietic anemia
Specific but not very sensitive.
Diagnostic Test
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The sugar water or sucrose lysis test uses the ionic
strength of serum that is reduced by adding an isoosmotic solution of sucrose, which then activates the
classic pathway: sucrose molecules enter red cells
through defects and produce osmotic lysis.
PNH diagnosed by >5% lysis.
Less specific but more sensitive.
Diagnostic Test
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Low-tech test. The
patient is asked to
collect a sample of
urine each hour for 48
hours. The physician
lines them up and eyes
their colors, and the
diagnosis is plain.
Diagnostic Test
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Flow Cytometry
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Expression of GPI-anchored proteins CD55 and
CD59 analyzed on hematopoietic cells using
monoclonal antibodies and flow cytometry.
Highly specific. No other condition in which red
cells are a mosaic of normal and GPI linked
protein deficient cells.
Flow Cytometric Analysis
Hillmen et al, NEJM, 1995
Pathogenesis
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Relative/absolute bone marrow failure
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Two stage model
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present to some degree in all patients
relative granulocytopenia/thrombocytopenia
decreased capacity to form myeloid colonies
somatic mutation in PIG-A gene (understood)
some cause for bone marrow failure (not understood)
Is damage directed at a GPI linked molecule?
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Mutant clone may expand as a result of an immuneescape from antigen-driven lymphocyte attack on
hematopoietic progenitors.
Dual Pathogenesis Hypothesis
Hillmen and Richards, Br J Haematol, 2000
Aplastic Anemia and PNH
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The association between PNH and aplastic anemia
goes both ways.
Although current emphasis is on patients with
marrow failure who are found to have PNH clones,
the older literature describes PNH patients with
apparent progression to aplastic anemia. In such
instances, the endstage has been called "spent
PNH“.
All four of the following clinical scenarios are based
on the same pathophysiologic mechanisms. The
only difference is the temporal relationship between
bone-marrow failure and somatic PIG-A mutation.
Aplastic Anemia and PNH
Natural History of PNH
Hillmen et al, NEJM, 1995
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Long term study of 80 patients with PNH seen at one institution
between 1940 and 1970
Results
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median age at diagnosis: 42 (16-75)
median survival: 10 years
28% survived more than 25 years
39% had one or more episodes of venous thrombosis
12 experienced spontaneous clinical recovery
leukemia did not develop in any of the patients
Sites of Thrombosis in PNH
Hillmen et al, NEJM, 1995
Natural History of PNH
Hillmen et al, NEJM, 1995
Prognostic Factors
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Thrombosis
Evolution to pancytopenia, myelodysplastic syndrome, or
acute leukemia (1% ~ 10-100x more than normal)
Age >55
Evidence of deficient hematopoiesis at disease onset,
such as aplastic anemia or thrombocytopenia
Treatment
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Supportive, prevent complement activation
 Prednisone beneficial. Moderate doses (15-30 mg)
administered on alternate days. Higher doses for acute
episodes.
 Fe supp given urinary loss (10-20x normal). Suppress
hemoglobinuric episode with prednisone.
 Folic Acid supp given increased need of hyperplastic
marrow for cofactor.
 pRBC transfusion as needed.
 Androgenic hormones effective but mechanism unclear.
 Eculizumab a monoclonal Ab that binds to C5 component
of complement and inhibits terminal complement activation
being studied.
Treatment
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Therapy for/prevention of thrombosis
 Thrombolytics acutely
 Anticoagulation
 First episode - managed as other patients with similar event
 Recurrent episodes – lifetime
 Prophylaxis –
 Retrospective studies only, suggest warfarin prophylaxis
effective in patients with PNH if the granulocyte clone size is
>50%, platelet count >100K, no contraindications to ac.
 Heparin or LMWH should be used in any perioperative
period, during immobilization, or with use of indwelling
intravenous catheter. Also start in 1st trimester of pregnancy
until 4-6 weeks post-partum.
 Efficacy of anti-platelet agents not clear.
Treatment
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Stimulate hematopoiesis
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G-CSF
Immunosuppression - hypothesis that immune-mediated
bone marrow damage in PNH is primarily directed
against the normal GPI-positive cells, producing growth
advantage for PNH cells. Improved impaired
hematopoiesis, but hemolysis and PNH clone not
affected.
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ATG
Cyclosporine
Treatment
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Protein transfer – Transfer of GPI-linked proteins
feasible using either high density lipoproteins or
washed RBC microvesicles. PNH cells show
increased cell-associated CD55 and CD59 levels and
decreased hemolysis.
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Gene Therapy - PIG-A gene cloned. But PIG-A
inactivation alone does not confer a proliferative
advantage to hematopoietic stem cell. Correcting the
PNH defect may allow exposure to the insult causing
bone marrow failure.
Treatment
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Stem Cell Transplantation
Indications: severe bone marrow hypoplasia, severe thrombotic
events (hepatic vein), children
 Syngeneic (identical twin) – shown to be successful
 Autologous - not very successful due to inability to obtain
sufficient numbers of normal cells
 Allogeneic
 Allogeneic hematopoietic cell transplantation (HCT) after highdose conditioning is the only curative treatment; however, it is
associated with high treatment-related mortality.
Circulating Stem Cells in PNH
Johnson et al, Blood, 1998
Stem Cell Transplantation in
PNH
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Summary of single institution trials
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Approximately 12 reported
Number of patients ranges from 1-16
Survival rates typically higher (58-100%)
Likely high degree of reporting bias, small studies
Stem Cell Transplantation in
PNH
IBMTR Data
Saso et al, Br J Haematol, 1999
Results
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Sustained engraftment: 77%
Graft failure: 17%
Grade 2-4 acute GVHD: 34%
Chronic GVHD: 33%
Causes of death:
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graft failure (7), int. pneumonitis (4), GVHD (3),
infection (3), ARDS (2), hemorrhage (1)
IBMTR Data
Saso et al, Br J Haematol, 1999
Stem Cell Transplantation in
PNH
Matched siblings
IBMTR Data
Saso et al, Br J Haematol, 1999
Stem Cell Transplantation in
PNH
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Conclusions from reported series:
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BMT may cure 50-60% of selected patients with
HLA-identical siblings
Most patients transplanted have been < 30 years
of age
Regimen related toxicity and GVHD remain
significant hurdles
Role of alternative donor transplants unclear,
though initial reports are not encouraging except
in pediatric population
Alternative Treatment
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http://www.herbchina2000.com/therapies/HP
H.shtml
Prognosis Based on
Management
Luzzatto, Haematologica, 2000
PNH: Management Guidelines
Luzzato, ASH, 2001
Case
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Started on prednisone, Fe, and Folate supplementation
by Hematologist.
One year after diagnosed with PNH, patient admitted to
PHD for pneumonia. WBC 2900 (ANC1800), H/H
11.7/33.6, plts 81,000 (labs one week prior normal CBC).
LDH 2147, Tbili 2, Retic 3%.
Obtained records (bone marrow results) revealing
diagnosis.
Prophylactic lovenox and higher dose prednisone given.
Blood counts improved.
Did well. Discharged to f/u with his Hematologist.
References
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Firkin, F, Goldberg, H, Firkin, BG. Glucocorticoid management of
paroxysmal nocturnal hemoglobinuria. Australia Ann Med 1968; 17:127.
Rosse, WF. Treatment of paroxysmal nocturnal hemoglobinuria. Blood
1982; 60:20.
Hartmann, RC, Jenkins, DE Jr, Mckee, LC, heyssel, RM. Paroxysmal
nocturnal hemoglobinuria: Clinical and laboratory studies relating to iron
metabolism and therapy with androgen and iron. Medicine 1966; 45:331.
Saso, R, et al. Bone marrow transplants for paroxysmal nocturnal
haemoglobinuria. Br J Haematol. 1999 Feb;104(2):392-6
Risitano AM, et al. Large granular lymphocyte (LGL)-like clonal expansions
in paroxysmal nocturnal hemoglobinuria (PNH) patients Leukemia. 2005
Feb;19(2):217-22
Raiola, AM, et al Bone marrow transplantation for paroxysmal nocturnal
hemoglobinuria.
Haematologica. 2000 Jan;85(1):59-62
References
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Moyo, VM, Mukhina, GL, Garrett ES, Brodsky, RA. Natural history of
paroxysmal nocturnal hemoglobinuria using modern diagnostic assays. Br J
Haematol 2004; 126:133.
Nishimura, J, Kanakura, Y, Ware, RE, et al. Clinical course and flow
cytometric analysis of paroxysmal nocturnal hemoglobinuria in the United
States and Japan. Medicine (Baltimore) 2004; 83:193.
Karadimitris, A, Luzzatto L. The cellular pathogenesis of paroxysmal
nocturnal haemoglobinuria. Leukemia. 2001 Aug;15(8):1148-52
Hillmen, P, et al. Natural History of Paroxysmal Hemoglobinuria. NEJM.
1995; 333 : 19.
Diagnosis and Treatment of paroxysmal nocturnal hemoglobinuria. UTD.
Clinical Manifestations of paroxysmal nocturnal hemoglobinuria. UTD.
http://hematology.im.wustl.edu/conferences/presentations/devine011703.
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http://www.path.sunysb.edu/labs/pnh/PNH_files/frame.
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References
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Luzzatto L, Araten DJ Allogeneic bone marrow transplantation for
paroxysmal nocturnal hemoglobinuria.
Haematologica. 2000 Jan;85(1):1-2
Research